CGRP通过ALKBH5/m6A抑制自噬促进缺氧/复氧心肌细胞增殖

The effect of CGRP on autophagy regulation through ALKBH5/m6A in hypoxia/reoxygenation-induced cardiomyocyte proliferation and apoptosis

目的探讨CGRP通过ALKBH5/m6A调控自噬对缺氧/复氧心肌细胞增殖和凋亡的影响。方法将心肌细胞H9c2随机分为Control组(H9c2细胞正常培养基培养)、H/R组(制备缺氧/复氧H9c2细胞损伤模型组)、CGRP组(缺氧/复氧模型前加入1×10^(-1)mol/L的CGRP作用20 min)和CGRP+sh-ALKBH5组(将sh-ALKBH5转染至H9c2细胞中,培养24 h后加入1×10^(-1)mol/L CGRP作用20 min,后制备缺氧/复氧模型)。采用CCK-8和EdU染色检测H9c2细胞活力;流式细胞仪检测细胞凋亡率;检测心肌细胞中乳酸脱氢酶(LDH)活性;Dot blot检测m6A甲基化水平;蛋白质印迹法和RT-PCR检测细胞中ALKBH5、Beclin-1、p62、LC3Ⅱ、LC3Ι蛋白和m RNA表达。结果与Control组比较,H/R组细胞存活率[(46.27±4.09)%]、EdU阳性细胞率[(3.89±0.87)%]明显降低,细胞凋亡率[(13.62±1.27)%]、LDH浓度(276.32±25.14)、m6A相对表达量(2.46±0.18)明显增加(P<0.05);与H/R组比较,CGRP组细胞存活率[(87.31±6.24)%]、EdU阳性细胞率[(40.71±2.53)%]均明显增加,细胞凋亡率[(3.17±0.82)%]、LDH浓度(78.24±7.19)、m6A相对表达量(1.42±0.13)明显降低(P<0.05);CGRP+sh-ALKBH5组细胞存活率[(61.04±5.48)%]、EdU阳性细胞率[(8.76±1.24)%]均明显低于CGRP组,细胞凋亡率[(10.19±0.95)%]、LDH浓度(229.06±22.43)、m6A相对表达量(2.09±0.15)明显高于CGRP组(P<0.05)。与Control组比较,H/R组细胞中ALKBH5(0.34±0.03)、p62蛋白(0.32±0.04)和mRNA表达(0.21±0.04)明显降低,Beclin-1蛋白(0.97±0.13)及mRNA表达(2.14±0.17)和LC3Ⅱ/LC3Ι比值(0.64±0.08)明显升高(P<0.05);与H/R组比较,CGRP组细胞中ALKBH5(0.78±0.09)、p62蛋白(0.71±0.08)和m RNA(0.87±0.08)表达明显增加,Beclin-1蛋白(0.41±0.06)及mRNA表达(1.26±0.12)和LC3Ⅱ/LC3Ι比值(0.23±0.03)明显降低(P<0.05);与CGRP组比较,CGRP+sh-ALKBH5组细胞中ALKBH5(0.50±0.07)、p62蛋白(0.40±0.05)和mRNA表达(0.30±0.05)明显降低,Beclin-1蛋白(0.86±0.10)及mRNA表达(1.95±0.14)和LC3Ⅱ/LC3Ι比值(0.58±0.06)明显升高(P<0.05)。结论CGRP可能通过促进m6A去甲基酶ALKBH5的表达来发挥作用,抑制心肌细胞在缺氧/复氧条件下的自噬和凋亡,从而提高心肌细胞的存活率。

Objective To explore the effect of CGRP on autophagy regulation through ALKBH5/m6A in hypoxia/reoxygenation-induced cardiomyocyte proliferation and apoptosis.Methods H9c2 cardiomyocytes were randomly divided into Control group(H9c2 cells cultured in normal culture medium),H/R group(preparation of hypoxia/reoxygenation-induced H9c2 cell injury model),CGRP group(1×10^(-1)mol/L CGRP added before hypoxia/reoxygenation for 20 min),and CGRP+sh-ALKBH5 group(sh-ALKBH5 transfected into H9c2 cells,cultured for 24 h,followed by the addition of 1×10^(-1)mol/L CGRP for 20 min,and then preparation of hypoxia/reoxygenation model).CCK-8 and EdU staining were used to detect cell viability of H9c2 cells;flow cytometry was used to measure cell apoptosis rate;lactate dehydrogenase(LDH)activity was measured in cardiomyocytes;Dot blot was performed to measure m6A methylation levels;protein immunoblotting and RT-PCR were performed to measure the expression of ALKBH5,Beclin-1,p62,LC3Ⅱ,LC3Ιproteins,and mRNA in cells.Results Compared with the Control group,the H/R group showed significantly decreased cell survival rate(46.27%±4.09%),EdU positive cell rate(3.89%±0.87%),and increased cell apoptosis rate(13.62%±1.27%),LDH concentration(276.32%±25.14%),and m6A relative expression level(2.46%±0.18%)(P<0.05).Compared with the H/R group,the CGRP group exhibited significantly increased cell survival rate(87.31%±6.24%),EdU positive cell rate(40.71%±2.53%),and decreased cell apoptosis rate(3.17%±0.82%),LDH concentration(78.24%±7.19%),and m6A relative expression level(1.42%±0.13%)(P<0.05).The cell survival rate(61.04%±5.48%)and EdU positive cell rate(8.76%±1.24%)in the CGRP+sh-ALKBH5 group were significantly lower than those in the CGRP group,while the cell apoptosis rate(10.19±0.95%),LDH concentration(229.06±22.43%),and m6A relative expression level(2.09±0.15%)were significantly higher than those in the CGRP group(P<0.05).Compared with the Control group,the ALKBH5 protein(0.34±0.03),p62 protein(0.32±0.04),and mRNA expression(0.21±0.04)in the H/R group were significantly reduced,while Beclin-1 protein(0.97±0.13),mRNA expression(2.14±0.17),and LC3Ⅱ/LC3Ιratio(0.64±0.08)were significantly increased(P<0.05).Compared with the H/R group,the CGRP group showed significantly increased ALKBH5 protein(0.78±0.09),p62 protein(0.71±0.08),and mRNA expression(0.87±0.08),while Beclin-1 protein(0.41±0.06),mRNA expression(1.26±0.12),and LC3Ⅱ/LC3Ιratio(0.23±0.03)were significantly decreased(P<0.05).Compared with the CGRP group,the CGRP+sh-ALKBH5 group exhibited significantly decreased ALKBH5 protein(0.50±0.07),p62 protein(0.40±0.05),and mRNA expression(0.30±0.05),while Beclin-1 protein(0.86±0.10),mRNA expression(1.95±0.14),and LC3Ⅱ/LC3Ιratio(0.58±0.06)were significantly increased(P<0.05).Conclusion CGRP can inhibit hypoxia/reoxygenation-induced autophagy and apoptosis in myocardial cells,increase cell survival rate,and its mechanism of action may be related to the promotion of m6A demethylase ALKBH5 expression.